The present invention relates to a process of producing and/or purifying virtually any hybrid polypeptide or fused protein molecule employing recombinant DNA techniques. More specifically, a DNA fragment coding for a protein molecule, e.g. a polypeptide or portion thereof, is fused to a DNA fragment coding for a binding protein such as the gene coding for the maltose binding protein. The fused DNA is inserted into a cloning vector and an appropriate host transformed. Upon expression, a hybrid polypeptide or fused protein molecule is produced which can be purified by contacting the hybrid polypeptide with a ligand or substrate to which the binding protein has specific affinity, e.g. by affinity chromatography. The hybrid polypeptide so purified may in certain instances be useful in its hybrid form, or it may be cleaved to obtain the protein molecule itself by, for example, linking the DNA fragments coding for the protein molecule and binding protein with a DNA segment which codes for a peptide which is recognized and cut by a proteolytic enzyme. The present invention also relates to certain vectors useful in practing the above process as well as to a bioreactor and methods employing the bound hybrid polypeptide, e.g. where the bound fused polypeptide is contacted and reacted with a susbstrate which interacts with the bound protein molecule to produce a desired result.
Recently developed techniques have made it possible to employ microorganisms, capable of rapid and abundant growth, for the synthesis of commercially useful proteins and peptides. These techniques make it possible to genetically endow a suitable microorganism with the ability to synthesize a protein or peptide normally made by another organism. In brief, DNA fragments coding for the protein are ligated into a cloning vector such as a plasmid. An appropriate host is transformed with the cloning vector and the transformed host is identified, isolated and cultivated to promote expression of the desired protein. Proteins so produced are then isolated from the culture medium for purification.
Many purification techniques have been employed to harvest the proteins produced by recombinant DNA techniques. Such techniques generally include segregation of the desired protein based on its distinguishing molecular properties, e.g. by dialysis, density-gradient centrifugation and liquid column chromatography. Such techniques are not universally applicable and often result in consumption of the purification materials which may have considerably more value than the protein being purified, particularly where substantial quantities of highly purified protein are desired.
Other procedures have been developed to purify proteins based on solubility characteristics of the protein. For example, isoelectric precipitation has been employed to purify proteins since the solubility of proteins varies as a function of pH. Similarly, solvent fractionatton of proteins is a technique whereby the solubility of a protein varies as a function of the dielectric constant of the medium. Solvent fractionation, while giving good yields often causes denaturation of the protein molecule. Neither isoelectrtc precipitation nor solvent fractionation are useful in obtaining highly purified protein. Such techniques are typically employed in tandem with other procedures.
Proteins have also been separated based on their ionic properties by e.g. electrophorests, ion-exchange chromatography, etc. Such electrophoretic techniques, however, have been used as analytical tools and are not practical as a means for purifying proteins on a large scale. Moreover, high purity and yield of the protein obtainable by such techniques is rarely achieved in a single step.
Affinity chromatography has also been employed in the purification of biopolymers such as proteins. Affinity chromatography involves a selective adsorbent which is placed in contact with a solution containing several kinds of substances including the desired species to be purified. For example, when used in protein purification protocols, affinity chromatography generally involves the use of a ligand which specifically binds to the protein to be purified. In general, the ligand is coupled or attached to a support or matrix and the coupled ligand contacted with a solution containing the impure protein. The non-binding species are removed by washing and the desired protein recovered by eluting with a specific desorbing agent. While affinity chromatography produces a relatively high leveI of purified protein, this technique requires significant amounts of the protein-specific ligand employed for purification. Moreover, the ligand will be different for each and every protein to be purified which necessarily entails a time-consuming and laborious regime. In addition, it has been found that specific ligands do not exist for all types of protein molecules, such as certain enzymes. As a result, affinity chromatography has not been successfully employed as a universal isolation purification technique for protein molecules.
One proposed attempt to universalire affinity chromatography to all proteins is described in European Patent Application 0,150,126 (Hopp). Disclosed is the preparation of a hybrid molecule produced by recombinant DNA techniques employing gene fusion. One gene codes for the desired protein to be purified while the other codes for an identification or marker peptide. The marker peptide contains a highly antigenic N-terminal portion to which antibodies are made and a linking portion to connect the marker peptide to the protein to be purified. The linking portion of the marker peptide is cleavable at a specific amino acid residue adjacent the protein molecule to be purified by use of a specific proteolytic agent. The fused or hybrid protein is isolated by constructing an affinity column with immobilized antibody specific to the antigenic portion of the marker peptide. The antibody binds to the fused protein which can thereafter be liberated from the column by a desorbing agent. The marker peptide may then be cleaved from the desired protein molecule with a proteolytic agent.
While purportedly overcoming some of the problems described above for protein purification protocols, Hopp requires substantial amounts of antibodies specific for the antigenic portion of the marker peptide. Moreover, the quantity of desorbing agent (in this case, a small peptide) required to compete off the target protein is substantial as well as a significant cost factor. Also, the desorbing agent must be purified away from the target protein. Thus, scale up for this system would not be practical. Furthermore, regeneration of the chromatographic column may be extremely difficult due to the destabilizing conditions employed to wash out the column after use, which may, in fact destroy the column. Others have suggested the use of low affinity antibody columns. However, low affinity columns often result in non-specific binding and would require significant cost for any large scale purification.
Thus, there is a continuing need for techniques which enable large scale purification of proteins produced through recombinant DNA processes without the above described problems. It would be particularly advantageous to provide an affinity purification process which utilizes an abundant and inexpensive ligand to which the fused protein would bind and an equally abundant and inexpensive desorbing agent.